These studies are designed to elucidate the enzymatic mechanisms of biosynthesis of the complex lipopolysaccharide of gram-negative bacteria, the mechanism of assembly of the outer membrane of which the lipopolysaccharide is a major constituent, and the factors responsible for the structural and functional integrity of the multienzyme system of O-antigen synthesis as it exists in the membrane. Studies on biosynthesis of lipopolysaccharide will focus on the internal Lipid A and backbone regions and will continue to emphasize the use of mutants to elucidate the pathways of biosynthesis. Newly developed techniques for separation of cytoplasmic and outer membrane will be employed to investigate the mechanism of assembly of the outer membrane and the role of the cytoplasmic membrane in this process. The structural and functional organization of the enzymes of O-antigen synthesis in the cytoplasmic membrane will be investigated by solubilization and purification of the enzymes and reconstitution of the multienzyme system from purified protein and lipid components.